Microscope illumination systems direct light onto a specimen and create "bright field" or "dark field" illumination. Bright field illumination is so named because light rays passing through the field surrounding the specimen and entering the microscope objective lens are unimpeded and thus bright compared to light rays attenuated by passing through the specimen. In contrast, in a "dark field" system, the relative brightness is reversed for the viewer by directing light rays onto the specimen field at an angle which falls outside the objective viewing aperture. The light passing through the specimen field surrounding the specimen is unimpeded and not observable through the objective lens of the microscope. However, some of the light directed onto the specimen is scattered and enters the viewing objective. Thus, the specimen appears brighter than the surrounding dark field.
Similarly, in fluorescence-based microscopy, light is typically directed into the specimen field at an angle which minimizes the amount of illumination light that enters the observation optics, for example it passes through the optics in a direction opposite to the light path of the observer. Use of filters to remove remaining stray illumination and scattered light makes the field appear dark to an observer. Excitation of fluorophores in the fluorescent sample give off secondary light, making the specimen appear bright. In a typical application, the illumination light passes through the specimen only once.
One use for fluorescence-based microscopy is the detection and quantification of inorganic, organic and biological polymers. Fluorescence is analyzed in clinical settings to obtain measurements in connection with immunology, toxicology, microbiology, drug screening, clinical chemistry, histopathology, and the like. Fluorescence is analyzed in many contexts to study enzymes, amino acids, carcinogens, and a wide variety of other chemical compounds. Nucleic acids such as DNA and RNA, proteins, chromosomes and other macromolecular structures are all visualized by fluorescence-based microscopy. Arrays of biological polymers are monitored by fluorescence-based microscopy for nucleic acid sequencing by hybridization, detection of genetic polymorphisms, drug screening and many other uses. For instance, comparative genomic hybridization (CGH) is a well-known approach for identifying the presence and localization of amplified or deleted sequences in a genome compared to a reference genome. See, Kallioniemi, et al. (1992) Science 258:818 and Pinkel et al. PCT/US95/16155 (WO 96/17958). CGH reveals amplifications and deletions irrespective of genome rearrangement and is used, e.g., for cancer assessment and diagnosis by monitoring amplifications or deletions associated with various cancers. CGH can provide a quantitative estimate of copy number and also provides information regarding the localization of amplified or deleted sequences in a normal chromosome.
Another increasingly useful florescence-based technology provides high density arrays of biological polymers on substrates, typically hundreds to thousands to tens of thousands of distinct polymers per square cm. This permits screening of thousands of different molecular interactions simultaneously. For example, very large scale immobilized polymer arrays (VLSIPS.TM. arrays) are used for the detection of nucleic acids for a variety of purposes. See, Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719 and Kozal et al. (1996) Nature Medicine 2(7): 753-759. See also, Pinkel et al. PCT/US95/16155 (WO 96/17958). Analysis of these arrays requires high sensitivity quantitative fluorescence measurements covering areas of typically one to several cm.sup.2.
Conventional fluorescence microscopes and laser scanning microscopes are suitable for such measurements, but they acquire results slowly because they only examine a small region on the substrate at one time. Conventional microscopes are also limited by the passage of excitation light through the same lens that collects the fluorescence for the specimen. This produces autofluorescence in the lens that interferes with the ability to observe weak signals.
The present invention solves these and other problems by providing apparatus and methods for illuminating large areas at one time such that illumination light makes multiple passes through the specimen, thereby increasing signal intensity. The illumination light does not enter the observation optics, minimizing optical "noise" in the system.